The government may have certain rights in this invention, since it was supported in part by a research grant awarded by the National Institutes of Health.
The blood clotting system is a natural and necessary process which occurs in humans and other vertebrates to seal the circulatory system from blood loss upon injury. The thrombus, or blood clot, forms from a complex cascade of reactions involving a number of plasma glycoproteins. In one of the final steps of clot formation, thrombin is formed by cleavage of prothrombin, which reaction is catalyzed by activated Factor X (Factor X.sub.a). Activated Factor V (Factor V.sub.a), Ca.sup.2+ and phospholipid membranes enhance the activity of Factor X.sub.a in activating prothrombin. Thrombin catalyzes formation of fibrin (which polymerizes to form a soft clot) from fibrinogen.
Thrombin catalyzes proteolysis of Protein C which inactivates Factor V.sub.a. This feedback mechanism is important because clot formation must be limited in vivo to prevent clots from forming in areas where the body not only does not need them, but where they can possibly cause injuries, such as stroke, heart attack, spontaneous abortions and other thrombotic diseases. These injuries often cause fatalities.
Protein C is a vitamin K-dependent protein plasma zymogen of the anticoagulant serine protease, activated Protein C (also referred to herein as "APC"). Protein C is activated primarily on the surface of endothelium by a complex composed of thrombin and thrombomodulin. Thrombomodulin acts to specifically bind thrombin. Thrombin bound to thrombomodulin thereby obtains a diminished ability to cause clot formation. Thrombin bound to thrombomodulin also obtains increased capacity to activate Protein C. Activated Protein C then functions as an anticoagulant by proteolytically inactivating Factors V.sub.a and VIII.sub.a, a process that is enhanced by a vitamin K dependent cofactor, protein S, negatively charged membrane surfaces and Ca.sup.2+ ions.
One group of patients who are at increased risk for thrombotic diseases are those who have lupus anticoagulants, which are antibodies which bind to anionic phospholipids used in clotting assays based on the PTT (partial thromboplastin time) or APTT (activated partial thromboplastin time) techniques. See The Merck Manual (16th Ed. 1992) at 1225; J. E. Ansell, Handbook of Hemostasis and Thrombosis (Little, Brown & Co., Boston) at 19 (1986). Typical PTT test results for patients having the lupus anticoagulant are a prolonged clotting time that fails to correct with a 1:1 mixture of the patient's and normal plasma, a normal or minimally prolonged PT (prothrombin time), and a nonspecific depression of those clotting factors measured by a PTT technique (Factors XII, XI, IX and VIII). The lupus anticoagulant antibodies may also react with cardiolipin which can interfere with assays utilizing cardiolipin as a reagent. See The Merck Manual, supra. Anti-cardiolipin or anti-phosphatidylethanolamine antibodies can cross react with each other, but not interact with sufficient affinity to procoagulant phospholipids to be anticoagulants. Because of the specificity of phospholipids for activated Protein C, such antibodies would selectively inhibit activated Protein C anticoagulant activity without influencing the coagulation tests performed in the absence of activated Protein C which are used to diagnose the presence of a "lupus anticoagulant."
Despite interference of the lupus anticoagulant antibodies with procoagulant phospholipid in clotting tests in vitro, persons with the antibodies have been reported to have an increased risk for thrombosis, either venous or arterial. Further, repeated spontaneous abortions in the first trimester of pregnancy have also been reported. Id. Patients have been treated with long term anticoagulant therapy to reduce the possibility of thrombosis, but no adequate technique has been developed for monitoring the effectiveness of such therapy. It should also be noted that other patients, who do not necessarily test positively for the lupus anticoagulant, may also be at risk for thrombotic disease. Further, not all persons who have the lupus anticoagulant or other risk factors may have the identical propensity for thrombosis.
In order to attempt to identify patients at risk for thrombosis, standard clotting tests have been performed on patient plasma. Additional testing has been suggested as described above where PTT and/or PT test results do not appear to be normal, such as repeating the test with added normal plasma. However, no technique has been developed to differentiate among lupus patients and among others which patients have the highest propensity to have a thrombotic incident.
In several clotting assays, it has been found beneficial to employ a membrane source to facilitate the reaction. It has been reported that the activity of Factor X.sub.a in activating prothrombin is enhanced many fold in the presence of Factor V.sub.a, Ca.sup.2+ and phospholipid membranes containing phosphatidylserine.
Analysis of the prothrombin activation complex, referred to as prothrombinase, has led to a model in which membrane acceleration of coagulation reactions is considered to be mediated by binding the enzyme, cofactor and substrate to the membrane surface, thereby increasing the local concentration of reactants and enhancing key protein-protein interactions-necessary for optimal catalysis. K. G. Mann et al., Ann Rev. Biochem. 57:915-56 (1988). This model is consistent with that proposed for the Factor V.sub.a inactivation complex. F. J. Walker, J. Biol. Chem. 256:11128-31 (1981). All studies have demonstrated that high affinity binding of these proteins involves negatively charged phospholipid surfaces. G. L. Nelsestuen et al., Biochemistry 16:4164-77 (1977); G. L. Nelsestuen et al., Biochemistry 17:2134-38 (1978); and R. A. Schwalbe et al., J. Biol. Chem. 264:20288-96 (1989). Of the naturally occurring negatively charged phospholipids, phosphatidylserine (also referred to herein as "PS") is the most effective functionally in the prothrombinase complex and in terms of binding interactions with Factors V.sub.a and X.sub.a. G. Pei et al., J. Biol. Chem. 268:3226-33 (1993). Taken together, these observations have led most investigators to assume that all of the complexes would share similar phospholipid requirements for optimal function. This assumption gained further support from the fact that .gamma.-carboxyglutamate (Gla) containing regions of the vitamin K dependent proteins share considerable sequence identity (P. Fernlund et al., J. Biol. Chem. 257:12170-79 (1982)), and it is this domain that is primarily responsible for membrane interaction. Schwalbe et al., supra. Previous investigators have thus assumed that the membrane requirements for inactivation of Factor V.sub.a with activated Protein C would be similar to those required for prothrombinase activity for activation of thrombin.
Surprisingly, it has now been found that phosphatidylethanolamine (also referred to herein as "PE") incorporation into membrane vesicles dramatically enhances APC inactivation of Factor V.sub.a without influencing prothrombinase activity. It has also now been found that patient plasma may be screened by assaying it in a one stage assay in the presence and absence of activated Protein C with a membrane source comprising an effective amount of PE. In employing PE in such assays, patient plasma containing lupus anticoagulants has been found to exhibit prolonged clotting times relative to normal pooled plasma control in the absence of activated Protein C, but to exhibit a clotting time less than normal control plasma in the presence of activated Protein C at appropriate phospholipid concentrations. Thus, by altering the composition of the membrane used in assays, recognition of patients most at risk for thrombosis can be determined and the effectiveness of antiinflammatory drugs used to reduce antibody titers, such as for example Prednisone (17, 21-Dihydroxypregna-1,4-diene-3,11,20-trione), or anticoagulation therapies can be monitored.